Cellular internalization of pIgR stalk and associated ligands

ABSTRACT

Compositions and methods for specific binding to the stalk of a polymeric immunoglobulin receptor (pIgR) of a cell with the proviso that the ligand does not substantially bind to secretory component of pIgR under physiological conditions. The ligand may be targeted to, into, or across the cell and may comprise a biologically active composition.

GOVERNMENT RIGHTS

This invention was made with Government support under Grant No. AI25144,awarded by the National Institutes of Health. The Government has certainrights in this invention.

This application claims the benefit of U.S. Provisional Application No.60/018,958, filed Jun. 4, 1996, the disclosure of which is incorporatedby reference.

RELATED APPLICATIONS

No related applications.

FIELD OF THE INVENTION

The present invention relates, in general, to compositions and methodsfor the specific binding of a ligand to the stalk region of thepolymeric immunoglobulin receptor for internalization into, or transportacross, a cell.

BACKGROUND OF THE INVENTION

One of the most challenging problems facing the pharmaceutical andbiopharmaceutical industries is delivering therapeutic agents past thevarious semi-permeable membranes within the body. Particularly in thecase of macromolecules, the obstacle to cost effective or convenienttreatment is often due to the lack of an adequate drug delivery system.In turn, this issue dictates whether production of a drug iseconomically feasible. Thus the search for alternative delivery systemsoften rivals the search for new drugs themselves.

Gene transfer methods can be viewed as a paradigm of macromolecular drugdelivery. These methods can be divided into three categories: physical(e.g., electroporation, direct gene transfer, and particle bombardment),chemical (e.g., proteinoids, microemulsions, and liposomes), andbiological (e.g., virus-derived vectors, and receptor-mediated uptake).Amongst biological transfer methods, receptor-mediated uptake is aparticularly promising approach. Targeting a ligand to an endocytosedreceptor acts as a means to ferry that ligand into the cell. However,one drawback of receptor-mediated systems has been their generalreliance on intravenous administration which severely limits their use.

Mucosal epithelial cells line a number of readily accessible tissuessuch as those found in the upper respiratory and gastrointestinaltracts. The accessibility of these cells make them an attractive targetfor drug delivery. See, e.g., Ferkol et al., J. Clin. Invest.92:2394-2400 (1993); Ferkol et al., J. Clin. Invest. 95:493-502 (1995).Retrograde transport of an antibody from the lumenal to the basolateralsurface of epithelial cells has been reported, albeit at very lowlevels. Breitfeld et al., J. Cell Biology 109:475-486 (1989). In thatstudy, movement across the cell was followed by binding an antibody tothe secretory component of polymeric immunoglobulin receptor (pIgR).Relative to the level of basolateral to apical transport, Breitfeld etal. reported that less than 5% of the transport was retrograde innature. The nominal level of counter-transport minimizes the utility ofsecretory component as a means to deliver biologically activecompositions into cells. Moreover, due to the abundance of cleaved pIgRin the lumen, binding of ligand to cleaved pIgR, rather than the intactpIgR of the cell surface, would diminish the utility of pIgRcounter-transport as a mechanism of drug delivery.

Accordingly, what is needed in the art is a means to convey ligands intoor across a cell surface with high efficiency. More particularly, whatis needed in the art is a means to deliver macromolecules to, into, oracross cells lining the gastrointestinal or respiratory tracts. Thepresent invention provides these and other advantages.

SUMMARY OF THE INVENTION

In one aspect, the present invention is directed to a ligand that bindsspecifically to the stalk of a polymeric immunoglobulin receptor (pIgR)of a cell with the proviso that the ligand does not substantially bindto secretory component of pIgR under physiological conditions.Typically, the antibody specifically binds only to the stalk. In oneembodiment, the ligand is an antibody, preferably a humanized antibody.In another embodiment the ligand is a recombinant single chain variableregion fragment of an antibody. In yet another embodiment the ligandbinds to an extracellular epitope within the first 33 amino acids thatare cell membrane proximal to a cleavage site of the receptor.

The ligand may comprise a binding component for binding to the stalk,and a biologically active component such as a nucleic acid, protein,radioisotope, lipid, and carbohydrate. Biologically active componentscomprise anti-inflammatories, anti-sense oligonucleotides, antibiotics,and anti-infectives. In one embodiment, the biologically activecomponent is a nucleic acid encoding a wildtype cystic fibrosistransmembrane conductance regulator. In a preferred embodiment the cellis an epithelial cell, most preferably a mammalian epithelial cell.

In another aspect, the present invention is directed to a method ofintroducing a ligand into a cell expressing a polymeric immunoglobulinreceptor by attaching the ligand to the stalk of the polymericimmunoglobulin receptor of the cell with the proviso that the liganddoes not substantially bind to the secretory component of pIgR underphysiological conditions. In one embodiment, the ligand is attached tothe stalk at the apical surface of a cell; and in further embodiments,the ligand is transcytosed to the basolateral surface of the cell, andreleased from the stalk at the basolateral surface.

In a further aspect, the present invention relates to a method ofattaching a ligand to a cell expressing a polymeric immunoglobulinreceptor comprising the step of binding the ligand to a stalk of thereceptor with the proviso that the ligand does not substantially bind tosecretory component of pIgR under physiological conditions. In oneembodiment, the ligand is introduced into the cell after binding.Alternative embodiments of the present invention may be had by referenceto the various aspects of the present invention.

Amongst the various in vivo and in vitro utilities, the presentinvention may be used to transport therapeutic or diagnosticcompositions to, into, or across, mucosal epithelial cells. Thus, theinvention provides a highly efficient and convenient means to transfernucleic acids or proteins into epithelial cells.

DESCRIPTION OF THE PREFERRED EMBODIMENT

The present invention is directed to a ligand that binds specifically tothe stalk of a polymeric immunoglobulin receptor (pIgR) of a cell withthe proviso that the ligand does not substantially bind to secretorycomponent of pIgR under physiological conditions. The inventionprovides, inter alia, methods of attaching and introducing a ligand intoa cell expressing pIgR.

After transport to the apical surface of epithelial cells, the majorityof pIgR is cleaved and secretory component is released. We havediscovered that, surprisingly and unexpectedly, cleavage of intact pIgRin the lumen leaves a residual extracellular region of pIgR (i.e., the"stalk") intact; and further, the stalk remains accessible to bindingdespite the abundance of proteases typically present in lumenal milieu.The ability to bind, endocytose, and transcytose a ligand bound to thepIgR stalk provides, in part, the invention as disclosed and claimedherein.

The present invention has utility as a means of transporting therapeuticor diagnostic compositions to, into (endocytosis) or across(transcytosis) a cell expressing pIgR. Thus the invention can be used totransport biologically active compositions such as proteins, nucleicacids, or detectable labels specifically to cells expressing pIgR. Theinvention also provides a means of labeling and distinguishingepithelial cells from amongst a mixed cell population in pathologicalstudies. Further, since pIgR expression is reduced in carcinomasrelative to normal epithelium, the labeling of pIgR has utility as adiagnostic adjunct in endoscopic or radiologic procedures. Additionally,binding of therapeutic ligands to pIgR has utility in extending theirduration in the lumen of various passageways and increasing theireffectiveness.

Definitions

Unless defined otherwise herein, all technical and scientific terms usedherein have the same meaning as commonly understood by one of ordinaryskill in the art to which this invention belongs. Singleton et al.(1994) Dictionary of Microbiology and Molecular Biology, second edition,John Wiley and Sons (New York), and Hale and Marham (1991) The HarperCollins Dictionary of Biology, Harper Perennial, N.Y. provide one ofskill with a general dictionary of many of the terms used in thisinvention. Amino acids may be referred to herein by either theircommonly known three letter symbols or by the one-letter symbolsrecommended by the IUPAC-IUB Biochemical Nomenclature Commission.Nucleotides, likewise, may be referred to by their commonly acceptedsingle-letter codes. For purposes of the present invention, thefollowing terms are defined below.

By "ligand" or "ligand binding moiety", is meant all molecules capableof specifically binding to the polymeric immunoglobulin receptor (pIgR).Ligands include, but are not limited to, antibodies, proteins, peptides,nucleic acids, lipids, and carbohydrates.

By "biologically active component" is meant a compound which, in vivo,directly causes or inhibits an increase or decrease in cellulartranscription, translation, receptor binding, active or passivetransport, cell signaling, signal transduction, cell division, celldifferentiation, cell death, cell adhesion, cell movement, cellmorphology, metabolism, enzyme activity, apoptosis, protein degradation,protein movement (e.g., secretion), protein stability, orphosphorylation. Biologically active components also comprise diagnosticcompositions which allow the foregoing events to be assessed.

By "bind(s) specifically" or "specifically bind(s)" or "attached" or"attaching" is meant the preferential association of a ligand, in wholeor part, with a cell or tissue bearing a particular target molecule ormarker and not to cells or tissues lacking that target molecule. It is,of course, recognized that a certain degree of non-specific interactionmay occur between a molecule and a non-target cell or tissue.Nevertheless, specific binding, may be distinguished as mediated throughspecific recognition of the target molecule. Typically specific bindingresults in a much stronger association between the delivered moleculeand cells bearing the target molecule than between the bound moleculeand cells lacking the target molecule. Specific binding typicallyresults in greater than 2 fold, preferably greater than 5 fold, morepreferably greater than 10 fold and most preferably greater than 100fold increase in amount of bound ligand (per unit time) to a cell ortissue bearing the target molecule as compared to a cell or tissuelacking the target molecule or marker.

By "stalk" is meant the extracellular component of the polymericimmunoglobulin receptor (pIgR) that corresponds to that region of pIgRthat is bound to the cell following cleavage of that segment of pIgRwhich constitutes the secretory component. The stalk is presentregardless of whether the segment of pIgR which corresponds to secretorycomponent is cleaved or uncleaved from pIgR.

By "pIgR" or "polymeric immunoglobulin receptor" is meant the receptorwhich is expressed in mucosal epithelial cells, including airwayepithelial cells, submucosal gland cells, intestinal cells, nasalepithelium, breast, oral mucosa, urinary and reproductive tractepithelium, and conjunctival tissue, and is implicated in basolateral toapical transcytosis of dimeric immunoglobulin A (digA) and/or pentamericIgM.

By "not substantially bind" is meant that no more than 15% of a ligandwhich specifically binds to a target molecule is bound to a particularnon-target molecule. More preferably, no more than 10% is bound to thenon-target molecule, even more preferably less than 5%, and mostpreferably less than 1%.

By "secretory component" is meant that extracellular portion of pIgRwhich is generally cleaved following basolateral to apical transcytosis.Typically, the secretory component comprises the dimeric IgA (dIgA)binding portion of pIgR. Secretory component is typically released intothe lumen with or without dIgA bound to the secretory component.

By "physiological conditions" is meant an extracellular milieu havingconditions (e.g., temperature, pH, and osmolarity) which allows for thesustenance or growth of a cell of interest.

By "antibody" is meant an immunoglobulin molecule obtained by in vitroor in vivo generation of the humoral response, and includes bothpolyclonal and monoclonal antibodies. The term also includes geneticallyengineered forms such as chimeric antibodies (e.g., humanized murineantibodies), heteroconjugate antibodies (e.g., bispecific antibodies),and recombinant single chain Fv fragments (scFv). The term "antibody"also includes antigen binding forms of antibodies (e.g., Fab, F(ab)₂).

By "humanized antibody" is meant an antibody which comprises a non-humanamino acid sequence but whose constant region has been altered to reduceimmunogenicity in humans.

By "wildtype cystic fibrosis transmembrane conductance regulator" ismeant a functional form of the cystic fibrosis transmembrane conductanceregulator (CFTR). Riordan et al., Science, 245:1066-1073 (1989).

By "apical surface" is meant that surface of a cell to which intact pIgRis transcytosed to after endocytosis from the basolateral surface.Generally, the apical surface of the cell adjoins a lumen and thereinintact pIgR is cleaved to release the secretory component.

By "basolateral surface" is meant that surface of a cell to which intactpIgR is delivered to after synthesis in the endoplasmic reticulum andpassage through the Golgi complex.

By "surface of the stalk" is meant the extracellular region of thestalk.

By "released" is meant the interference in the specific association of aligand, in whole or part, with its target molecule.

By "transcytosed" or "transcytosis" is meant conveyance from one plasmamembrane of the cell to another via an intracellular route. Typically,transcytosis occurs from the basolateral to apical or apical tobasolateral plasma membrane of the cell.

By "cell membrane proximal" is meant next to or nearer the cellmembrane.

By "extracellular" is meant the region extending outward from the lipidbilayer encompassing a cell.

Identification of pIgR

The nucleic acid and amino acid sequence of the polymeric immunoglobulinreceptor has been identified in a variety of taxonomically diversespecies. See, Piskurich et al., Journal of Immunology 154:1735-1747(1995). Identification of pIgR from other species can be accomplished byany number of methods well known to those of skill in the art. Forexample, using published pIgR sequences a nucleic acid probe to pIgR canbe constructed. The probe typically should be derived from a conservedregion of pIgR. Hybridization of the probe to a genomic or cDNA librarycan be used to identify pIgR in an unknown species. It will beunderstood by the skilled artisan that the nucleic acid sequence of thepIgR probe should generally be that of the species most closely relatedto the probed species. An extensive guide to the hybridization ofnucleic acids is found in Tijssen (1993) Laboratory Techniques inBiochemistry and Molecular Biology--Hybridization with Nucleic AcidProbes Part I, Chapter 2 "Overview of principles of hybridization andthe strategy of nucleic acid probe assays", Elsevier, N.Y.

In an alternative approach, pIgR or peptide fragments thereof (e.g.,secretory component) can be used to create antibodies to screenexpression libraries. See, e.g., Ferkol et al., J. Clin. Invest.95:493-502 (1995). These and other methods well known to the skilledartisan may be found, for example, in Berger and Kimmel, Guide toMolecular Cloning Techniques, Methods in Enzymology volume 152 AcademicPress, Inc., San Diego, Calif. (Berger); Sambrook et al. (1989)Molecular Cloning--A Laboratory Manual (2nd ed.) Vol. 1-3; and CurrentProtocols in Molecular Biology, F. M. Ausubel et al., eds., CurrentProtocols, a joint venture between Greene Publishing Associates, Inc.and John Wiley & Sons, Inc., (1994 Supplement) (Ausubel). Confirmationof the identity of a nucleic acid or protein as encoding pIgR may be hadby such approaches as constructing antibodies to the putative pIgRprotein and confirming the ability of these antibodies to bind to aprotein having the characteristics of pIgR (e.g., being present on thesurface of epithelial cells, binding of dimeric IgA or pentameric IgM,etc.).

Identification of the Stalk

The stalk can be identified by a variety of techniques well known tothose of skill. A putative heptapeptide consensus sequence whichidentifies the cleavage site of pIgR and thereby defines the aminoterminus of the stalk has been identified. The sequence Phe-Ala-Xaa-Glu(SEQ ID NO:1), where Xaa is a polar or charged amino acid, wasidentified as immediately preceding this putative cleavage site.Piskurich et al., Journal of Immunology 154:1735-1747 (1995). Cleavageat the consensus site liberates the secretory component and defines itscarboxy terminus. The carboxy terminus of secretory component may bealtered by secondary cleavage events (e.g., exopeptidase orendopeptidase activity) to yield secondary carboxy termini. Id. However,the amide linkage which initially defines the amino terminus of thestalk and the carboxy terminus of secretory component may be identifiedby sequence alignment and identification of the cleavage consensussequence.

Methods of alignment of sequences for comparison are well-known in theart. Optimal alignment of sequences for comparison may be conducted bythe local homology algorithm of Smith and Waterman (1981) Adv. Appl.Math. 2: 482; by the homology alignment algorithm of Needleman andWunsch (1970) J. Mol. Biol. 48: 443; by the search for similarity methodof Pearson and Lipman (1988) Proc. Natl. Acad. Sci. USA 85: 2444; bycomputerized implementations of these algorithms (including, but notlimited to CLUSTAL in the PC/Gene program by Intelligenetics, MountainView, Calif., GAP, BESTFIT, FASTA, and TFASTA in the Wisconsin GeneticsSoftware Package, Genetics Computer Group (GCG), 575 Science Dr.,Madison, Wis., USA); the CLUSTAL program is well described by Higginsand Sharp (1988) Gene, 73: 237-244 and Higgins and Sharp (1989) CABIOS5: 151-153; Corpet, et al. (1988) Nucleic Acids Research 16, 10881-90;Huang, et al. (1992) Computer Applications in the Biosciences 8, 155-65,and Pearson, et al. (1994) Methods in Molecular Biology 24, 307-31.Alignment is also often performed by inspection and manual alignment.

In another approach, secretory component can isolated from fluids in theapical lumen (e.g., milk or bile) and sequenced by amino acid sequencingmethods well known to those of skill such as Edman degradation, or massspectrometry. Eiffert et al., Hoppe-Seyler's Z. Physiol. Chem.365:1489-1495 (1984). Amongst the various secondary carboxyl endsdefined by secondary cleavage events, the carboxy terminal amino acidadjacent to the cleavage site can be identified.

Peptides which correspond to the pIgR stalk of selected speciesinclude:Mouse:Glu-Arg-Glu-Ile-Gln-Asn-Val-Arg-Asp-Gln-Ala-Gln-Glu-Asn-Arg-Ala-Ser-(SEQ IDNO:2)Gly-Asp-Ala-Gly-Ser-Ala-Asp-Gly-Gln-Ser-Arg-Ser-Ser-Ser-Ser-LysRat:Glu-Arg-Glu-Ile-Gln-Asn-Ala-Gly-Asp-Gln-Ala-Gln-Glu-Asn-Arg- (SEQ IDNO:3)Ala-Ser-Gly-Asn-Ala-Gly-Ser-Ala-Gly-Gly-Gln-Ser-Gly-Ser-Ser-LysHuman:Glu-Lys-Ala-Val-Ala-Asp-Thr-Arg-Asp-Gln-Ala-Asp-Gly-Ser- (SEQ IDNO:4)Arg-Ala-Ser-Val-Asp-Ser-Gly-Ser-Ser-Glu-Glu-Gln-Gly-Gly-Ser-Ser-ArgBovine:Glu-Ser-Val-Lys-Asp-Ala-Ala-Gly-Gly-Pro-Gly-Ala-Pro-Ala- (SEQ IDNO:5)Asp-Pro-Gly-Arg-Pro-Thr-Gly-Tyr-Ser-Gly-Ser-Ser-LysRabbit:Leu-Ala-Glu-Val-Ala-Val-Gln-Ser-Ala-Glu-Asp-Pro-Ala-Ser- (SEQ IDNO:6)Gly-Asp-Pro-Ala-Ser-Gly-Ser-Arg-Ala-Ser-Val-Asp-Ser-Gly-Ser-Ser-Glu-Glu-Gln-Gly-Gly-Ser-Ser-Arg-Ser-Lys

Cells Expressing pIgR

While the present invention broadly pertains to eukaryotic cells. ThepIgR expressing cell of the present invention is preferably a mammaliancell and more preferably a mammalian epithelial cell that normallysecretes IgA. Mammalian cells can be transfected with a nucleic acidencoding pIgR isolated, synthesized or otherwise derived from one ormore desired species. Methods of transfecting and expressing genes inmammalian cells are known in the art. Transducing cells with viralvectors can involve, for example, incubating viruses with cells withinthe viral host range under conditions and concentrations necessary tocause infection. See, e.g., Methods in Enzymology, vol. 185, AcademicPress, Inc., San Diego, Calif. (D. V. Goeddel, ed.) (1990) or M.Krieger, Gene Transfer and Expression--A Laboratory Manual, StocktonPress, New York, N.Y., (1990) and the references cited therein.

The culture of cells which can be used in the present invention includecell lines and cultured cells from tissue or blood samples is well knownin the art. Freshney (Culture of Animal Cells, a Manual of BasicTechnique, third edition Wiley-Liss, New York (1994)) and the referencescited therein provides a general guide to the culture of cells. Thenucleic acid sequences encoding pIgR from the desired species may beexpressed in a variety of eukaryotic host cells, including yeast, andvarious higher eukaryotic cells such as the COS, CHO and HeLa cellslines and myeloma cell lines as well as MDCK and human colon carcinomaderived cells such as Caco2. The recombinant protein gene will beoperably linked to appropriate expression control sequences for eachhost. For eukaryotic cells, the control sequences will include apromoter and preferably an enhancer derived, for example, fromimmunoglobulin genes, SV40, cytomegalovirus, etc., and a polyadenylationsequence, and may include splice donor and acceptor sequences.

Binding Ligands to the Stalk

The specific stalk binding ligand is not critical to this invention andvarious ligands may be used. A host of methods for construction andselection of ligands such as nucleic acids, proteins or peptides(collectively, "peptides), or antibodies, or small organics orinorganics (e.g., U.S. Pat. No. 5,143,854; WO 90/15070; WO 92/10092; WO96/11878) having the desired specific binding characteristics are wellknown in the art. Preferably, ligands of the present invention will,under physiological conditions, bind to the stalk without substantiallybinding to the secretory component of pIgR. More preferably, the ligandsof the present invention specifically bind only to the stalk underphysiological conditions. Typical physiological conditions vary fromtissue to tissue. However, exemplary physiological conditions areencountered in the gastrointestinal or respiratory tract of mammals,including but not limited to humans. A ligand may be chosen to bind toan extracellular ligand binding site ("epitope") contained within thefirst 6, 9, 12, 15, 18, 21, 24, 27, 30, or 33 membrane proximal aminoacids of the stalk.

Antibodies, including polyclonal, monoclonal, or recombinant singlechain Fv antibodies, can be constructed for use as ligands in thepresent invention. Methods of producing polyclonal and monoclonalantibodies are known to those of skill in the art. See, e.g., Coligan(1991) Current Protocols in Immunology Wiley/Greene, N.Y.; and Harlowand Lane (1989) Antibodies: A Laboratory Manual Cold Spring HarborPress, N.Y.; Stites et al. (eds.) Basic and Clinical Immunology (4thed.) Lange Medical Publications, Los Altos, Calif., and references citedtherein; Goding (1986) Monoclonal Antibodies: Principles and Practice(2d ed.) Academic Press, New York, N.Y.; and Kohler and Milstein (1975)Nature 256: 495-497; See, Huse etal. (1989) Science 246: 1275-1281; andWard, et al. (1989) Nature 341: 544-546. Birch and Lennox, MonoclonalAntibodies: Principles and Applications, Wiley-Liss, New York, N.Y.(1995).

Other suitable techniques for antibody or peptide ligand preparationinclude selection of libraries of recombinant antibodies/peptides inphage or similar vectors. High affinity antibodies and peptides to thestalk can be rapidly isolated by using phage display methods to expressrecombinant single chain Fv (scFv) fragments or peptide ligands on thephage surface. Briefly, genes encoding the surface protein of a phageare altered so as to allow the insertion of an antibody or peptide genewhich is expressed as a fusion protein on the surface of the phage thatcarries the gene. The phage expressing the desired antibody or peptideligand can be selectively enriched and isolated by virtue of itsaffinity/avidity for the stalk. The DNA encoding the ligand is packagedin the same phage and which allows the gene encoding the ligand to beisolated. A variety of such methods are amply discussed in theliterature and well known to the skilled artisan. See, e.g., Winter etal., Annu. Rev. Immunol. 12:433-455 (1994); Marks et al., J. Mol. Biol.222:581-597 (1991); Vaughan et al., Nature Biotechnology 14:309-314(1996), U.S. Pat. Nos. 4,642,334; 4,816,397; 4,816,567; 4,704,692; WO86/01533; WO 88/09344; WO 89/00999; WO 90/02809; WO 90/04036; EP 0 324162; EP 0 239 400.

In chemical peptide synthesis, a procedure termed "Divide, Couple andRecombine" (DCR) has been used to produce combinatorial peptidelibraries. See, Furka et al., Int. J. Pept. Protein Res. 37:487-493(1991) and Houghten et al., Nature 354:84-86 (1991). As an alternativeto DCR, peptide mixtures have also been made by direct coupling ofmonomer mixtures. See, Rutter et al., U.S. Pat. No. 5,010,175. The useof such methods to produce mixtures of other linear polymers, such as"peptoids", has been suggested. See, Simon, et al., Proc. Natl. Acad.Sci. USA 89:9367-9371 (1992). In oligonucleotide synthesis, "degenerate"or "wobble" mixtures of oligonucleotide products can be made by, forexample, delivery of equimolar mixtures of monomers to anoligonucleotide polymer at specific steps during synthesis. See,Atkinson and Smith, in "Oligonucleotide Synthesis. A PracticalApproach", 1984, IRL Press, Oxford, edited by M. Gait, pp 35-81. Thesemethods of synthesizing peptides or oligonucleotides provide largenumbers of compounds for testing which, if active, can be readilyidentified.

Preferably, ligands will be constructed to minimize immunogenicity inthe host as, for example, by maximizing the number of autologous (self)sequences present in the ligand. Accordingly, chimeric antibodies havingnon-xenogenic variable regions are preferred. Particularly preferred arethe use of antibodies in which xenogenic portions are excluded, or areessentially limited to the complementarity determining regions as inhumanized antibodies.

Ligand Binding and Testing

Binding (i.e., attachment) of the ligand to the stalk of pIgR may occurprior or subsequent to cleavage of secretory component; and the ligandmay be attached at the basolateral or apical surface. Thus, the ligandcan be endocytosed basolaterally or apically, or be subject to apical tobasolateral, or basolateral to apical transcytosis. The fate of theligand, or any element thereof, will vary according to itsphysico-chemical characteristics. Accordingly, the properties of theligand may be selected or designed to perform the desired function atthe cell surface, within the endosome, or following transcytosis. Forexample, varying the sensitivity of a ligand to proteolytic or reducingenvironments can be used to determine the distribution of ligand bound,internalized, or transported across the cell. Where desirable, a ligandmay be designed to remain specifically bound to the cell followingattachment or transcytosis or, alternatively, to be released into theextracellular milieu. Thus, the properties of any of the variouselements of the ligand, including the binding component, biologicallyactive component or linker, may be designed or selected to allow fordifferent degrees of affinity, stability, or activity at differentintracellular compartments or surfaces of the cell, as desired.

A. Ex Vivo Testing of Ligand Binding

In vitro binding of the ligand to the stalk may be conveniently assessedby measuring endocytosis or transcytosis of bound ligand in mammalianepithelial cells. "Endocytosis" refers generally to the phenomenon of acell ingesting material, e.g., by phagocytosis or pinocytosis.Receptor-mediated endocytosis provides an efficient means of causing acell to ingest material which binds to a cell surface receptor. See, Wuand Wu (1987) J. Biol. Chem. 262:4429-4432; Wagner et al. (1990) Proc.Natl. Acad. Sci. USA 87:3410-3414, and EP-A1 0388 758. Any number ofwell known methods for assaying endocytosis may be used to assessbinding. For example, binding, transcytosis, and internalization assaysare described at length in Breiftfeld et al. J. Cell Biol. 109:475-486(1989).

Apical endocytosis is conveniently measured by binding a ligand such asa Fab fragment to the stalk at the apical surface of Madin-Darby caninekidney (MDCK) cells at 4° C., warming to 37° C. for brief periods (0-10min), and cooling the cells back down to 4° C. Methods of pIgRexpression in MDCK cells is well known in the art. Breitfeld et al.,Methods in Cell Biology 32:329-337 (1989). Fab remaining on the surfaceare removed by stripping at pH 2.3. Intracellular Fab are those thatremain cell-associated after the stripping, while surface-bound Fab arethose removed by the acid wash. Controls for non-specific stickinginclude using pre-immune Fab and/or MDCK cells that are not transfectedwith pIgR.

Transcytosis can be readily assessed by allowing MDCK cells to bind theFab at the apical surface at 4° C., warming up to 37° C. for 0-240 min,and then measuring the amount of Fab delivered into the basolateralmedium. This basolaterally-delivered Fab is compared to the sum of Fabthat remains associated with the cells (intracellular or acid-stripped)and the Fab released back into the apical medium. Alternatively,transcytosis can be assessed by continuously exposing cells to the Fabin the apical medium and measuring accumulation of Fab in thebasolateral medium. This method avoids cooling the cells, but does notprovide the kinetics of transporting a single cohort of ligand. In bothmethods degradation of the Fab can be assessed by running aliquots ofthe transcytosed Fab on SDS-PAGE and probing a Western with antibodies.Non-specific transport (e.g. due to fluid phase endocytosis andtranscytosis, or paracellular leakage between cells) can be controlledfor by using MDCK cells that are not transfected with the pIgR and/orpre-immune Fab.

B. In Vivo Testing of Ligand Binding

Transcytosis in vivo may conveniently be assessed using pathogen-freeexperimental animals such as Sprague-Dawley rats. Labelled ligand (e.g.,radioiodinated antibody) can be administered into the nares. As will beunderstood by those of skill in the art, a "label" is a compositiondetectable by spectroscopic, photochemical, biochemical, immunochemical,electromagnetic, radiochemical, or chemical means such as fluorescence,chemifluoresence, or chemiluminescence. Apical to basolateraltranscytosis can be readily determined by measuring delivery of theligand into the circulation as determined by the presence of label. Theintegrity of the ligand recovered from the circulation can be assessedby analyzing the ligand on SDS polyacrylamide gel electrophoresis.

Biologically Active Component

The biologically active component of the ligand may be covalently ornon-covalently bound to the ligand. For example, chelators may be usedto bind various isotopes, or nucleic acids may be bound to the ligandvia hydrogen bonds. Biologically active components may also beencompassed within emulsions, proteinoids, or liposomes. As thoseskilled in the art will understand, such structures may be linkedcovalently to the ligand via derivatized polar head groups or viamembrane integral proteins. Binding components of the ligand may alsocomprise the biologically active component.

Biologically active components comprise any number of compounds known tothose of skill as anti-inflammatories, cytokines, anti-infectives,enzyme activators or inhibitors, allosteric modifiers, or antibiotics.Thus, biologically active component includes such compounds as nucleicacids, proteins, peptides, amino acids or derivatives, glycoproteins,radioisotopes, lipids, carbohydrates, or recombinant viruses. The term"nucleic acid" refers to a deoxyribonucleotide or ribonucleotide polymerin either single- or double-stranded form, and unless otherwise limited,encompasses known analogues of natural nucleotides. Nucleic acidsinclude anti-sense nucleic acids, derivatized oligonucleotides forcovalent cross-linking with single or duplex DNA, triplex formingoligonucleotides, or nucleic acids encoding proteins or peptides.Nucleic acids also includes compositions for gene therapy such as thoseencoding for the wildtype cystic fibrosis transmembrane conductanceregulator.

Attaching Biologically Active Compositions to the Ligand

The procedure for attaching a biologically active component to a ligandwill vary according to the chemical structure of the component.Generally, the ligands will contain a variety of functional groups whichare available for reaction with a suitable functional group on abiologically active molecule to bind the agent thereto. Alternatively,the ligand and/or biologically active component may be derivatized toexpose or attach additional reactive functional groups. Thederivatization may involve attachment of any of a number of linkermolecules such as those available from Pierce Chemical Company, RockfordIll. A "linker" as used herein refers to a molecule used to join,covalently or non-covalently, the ligand and biologically activecomponent. Suitable linkers are well known to those of skill in the artand include, but are not limited to, straight or branched-chain carbonlinkers, heterocyclic carbon linkers, or peptide linkers. See, e.g.,Birch and Lennox, Monoclonal Antibodies: Principles and Applications,Chapter 4, Wiley-Liss, New York, N.Y. (1995); U.S. Pat. Nos. 5,218,112,5,090,914; Hermanson, Bioconjugate Techniques, Academic Press, SanDiego, Calif. (1996).

Where both molecules are polypeptides, the linkers may be joined to theconstituent amino acids through their side groups (e.g., through adisulfide linkage to cysteine). A bifunctional linker having onefunctional group reactive with a group on a particular biologicallyactive component, and another group reactive with a ligand, may be usedto form the desired conjugate. Alternatively, derivatization may involvechemical treatment of the component; e.g., glycol cleavage of the sugarmoiety of the glycoprotein antibody with periodate to generate freealdehyde groups. The free aldehyde groups on the antibody may be reactedwith free amine or hydrazine groups on an agent to bind the agentthereto. (See U.S. Pat. No. 4,671,958). Procedures for generation offree sulfhydryl groups on antibodies or antibody fragments are alsoknown (See U.S. Pat. No. 4,659,839). Many procedure and linker moleculesfor attachment of various compounds including radionuclide metalchelates, toxins and drugs to proteins such as antibodies are known.See, for example, European Patent Application No. 188,256; U.S. Pat.Nos., 4,671,958, 4,659,839, 4,414,148, 4,699,784; 4,680,338; 4,569,789;and 4,589,071; and Borlinghaus et al. Cancer Res. 47: 4071-4075 (1987)).

It is sometimes desirable to release the conjugated molecule when it hasreached a target site. Therefore, conjugates comprising linkages whichare cleavable in the vicinity of the target site may be used. Cleavingof the linkage to release the biologically active component from theantibody may be prompted by enzymatic activity or conditions to whichthe conjugate is subjected either inside the target cell or in thevicinity of the target site. When the target site is a tumor, a linkerwhich is cleavable under conditions present at the tumor site (e.g. whenexposed to tumor-associated enzymes or acidic pH) may be used. Use ofthe cis-aconitic acid spacer is useful for releasing biologically activecomponents in endosomes. Similarly, disulfide linkages are cleavable inthe reductive environment of the endosomes.

A number of different cleavable linkers are known to those of skill inthe art. See U.S. Pat. Nos. 4,618,492; 4,542,225, and 4,625,014. Themechanisms for release of an agent from these linker groups include, forexample, irradiation of a photolabile bond and acid-catalyzedhydrolysis. U.S. Pat. No. 5,141,648 discloses immunoconjugatescomprising linkers of specified chemical structure, wherein the linkageis cleaved in vivo thereby releasing the attached compound(radiotherapeutic agent, drug, toxin, etc.). The linker is susceptibleto cleavage at a mildly acidic pH, and is believed to be cleaved duringtransport into the cytoplasm of a target cell, thereby releasing thebiologically active compound inside a target cell. U.S. Pat. No.4,671,958 includes a description of immunoconjugates comprising linkerswhich are cleaved at the target site in vivo by the proteolytic enzymesof the patient's complement system. In view of the large number ofmethods that have been reported for attaching a variety ofradiodiagnostic compounds, radiotherapeutic compounds, drugs, toxins,and other components to ligands one skilled in the art will be able todetermine a suitable method for attaching a given component to a ligandof the present invention.

Egress of the Ligand from the Endosome

A number of methods well known to the skilled artisan may be used totransport ligand, or any portion thereof, out of the endosome.

A poly-L-lysine/nucleic acid complex bound to a ligand which bindsspecifically to the stalk can be used for efficient transfection. Ferkolet al., J. Clin. Invest., 92:2394-2400 (1993); and Ferkol et al., J.Clin. Invest., 95:493-502 (1995).

In another approach, poly-L-lysine can be linked, such as by geneticfusion or chemical linkers, to a ligand that binds specifically to thestalk of pIgR. In turn, this complex can be linked to defectiveadenovirus. Curiel and co-workers have demonstrated that naked plasmidDNA bound electrostatically to poly-L-lysine orpoly-L-lysine-transferrin which has been linked to defective adenovirusmutants can be delivered to cells with transfection efficienciesapproaching 90%. The adenovirus-poly-L-lysine-DNA conjugate binds to thenormal adenovirus receptor and is subsequently internalized byreceptor-mediated endocytosis. This approach has been used to obtain asmuch as a 1000-fold increase in expression of gene therapy vectors.Herpes viruses have similar properties. Curiel et al. (1991) Proc NatlAcad Sci USA 88:8850-8854; Cotten et al. (1992) Proc Natl Acad Sci USA89:6094-6098; Curiel et al. (1992) Hum Gene Ther 3:147-154; Wagner etal. (1992) Proc Natl Acad Sci USA 89:6099-6103; Michael et al. (1993) JBiol Chem 268:6866-6869; Curiel et al. (1992) Am J Respir Cell Mol Biol6:247-252, and Harris et al. (1993) Am J Respir Cell Mol Biol9:441-447); Gao et al. (1993) Hum. Gene Ther. 4:17-24; Curiel etaal.U.S. patent application Ser. No. 07/768,039.

In yet another approach using influenza virus, a hydrophobic peptide inthe hemagglutinin can act as a fusion peptide at low pH to effect fusionof the virus with the membrane of the endosome and delivering the virusinto the cytoplasm. This peptide has been used intransferrin/peptide/poly-L-lysine/DNA complexes for gene transfer usingthe transferrin receptor and substantially improved the efficiency ofexpression. Wagner et al., Proc. Natl. Acad. Sci USA 89:7934-7938(1992). This peptide can be engineered into a ligand for transport ofthe ligand, or a portion thereof, out of the endosome.

A further approach may employ ricin A. Ricin A chain is capable ofpenetrating out of endosome and into the cytosol. Beaumell et al., J.Biol. Chem. 268:23661-23669 (1993). A ligand of the present inventionmay be linked to ricin A, such as by genetic fusion or chemical linkers.

Although the present invention has been described in some detail by wayof illustration and example for purposes of clarity of understanding, itwill be obvious that certain changes and modifications may be practicedwithin the scope of the appended claims.

EXAMPLE 1

Example 1 describes a method of producing and assaying for chickenantibodies and Fab fragments which specifically bind to the rabbit pIgRstalk region.

The membrane-spanning segment of the rabbit pIgR begins at the Valineresidue at position 630. The sequence of the twenty four extracellularresidues of the rabbit pIgR that precede the membrane-spanning segmentis:607-AspProAlaSerGlySerArgAlaSerValAspAlaSerSerAlaSerGlyGlnSerGlySerAlaLys-629 (SEQ ID NO:7).

Two peptides were synthesized (Immuno-Dynamics, Inc., La Jolla, Calif.)representing the extracellular, membrane proximal 16 and 23 amino acidsof pIgR. A C-terminal cysteine was added for conjugation purposes.Peptide sequences (in single letter code) were: DPA SGS RAS VDA SSA SGQSGS AKC (SEQ ID NO:8) for the primary peptide; and the subsequencepeptide: ASV DAS SAS GQS GSA KC (SEQ ID NO:9). Half of each amount ofpeptide was conjugated to keyhole limpet hemocyanin (KLH) (byImmuno-Dynamics, Inc).

The KLH-conjugated peptides were sent to Lampire Biological Laboratories(Pipersville, Pa.) for production of chicken antibodies in two chickensper peptide. Lampire's standard protocol for chicken immunization wasfollowed by collection of pre-immune eggs and a pre-immune test bleed;intramuscular injection of 2 mg of peptide with Freund's completesuspension at project initiation; intramuscular injection of 0.5 mg ofpeptide with Freund's incomplete suspension week 1; intramuscularinjection of 0.25 mg of peptide with Freund's incomplete suspension week2; rest week 3; intramuscular injection of 0.25 mg of peptide withFreund's incomplete suspension week 4; rest week 5, and test bleed week6. Daily egg collection began around week 6 and monthly test bleeds werecollected. Eggs were delivered monthly.

Upon arrival, egg yolks were carefully separated from egg whites, andstored at 4° C. in 50-80 mis of basic buffer (0.01M sodium phosphate pH7.5, 0.1M NaCl, 0.01% azide) per egg yolk until processed for extractingchicken antibody ("IgY"). IgY was extracted from batches of stored eggyolks by a series of PEG precipitations followed by a series of ammoniumsulfate precipitations, according to the method of Polson et al. ImmunolCommun. 9:475 (1980)). Briefly, solid PEG (polyethylene glycol, MW 8000)was added to yolks in basic buffer to 3.5% by weight of PEG to volume ofdiluted yolk, and stirred at room temperature until dissolved. Thesolution was centrifuged at 14,000 g for 10 min at 20° C. and decantedthrough a funnel containing a loose layer of absorbent cotton gauze.More PEG was added to the clear filtrate for a final PEG concentrationof 12% to precipitate the IgY. After sedimenting the precipitate bycentrifuging at 14,000 g for 10 min at 20° C., the precipitate wasdissolved in 60 ml of basic buffer per yolk and an equal volume of 24%PEG in basic buffer was added to reform the precipitate. The precipitatewas centrifuged twice more at 14,000 g for 10 min at 20° C. to removeall residual PEG solution. Pellets were dissolved in 30 mls of basicbuffer per egg yolk, and the protein was precipitated in 50% saturated(NH4)₂ SO4 by slowly adding an equal volume of saturated (NH4)₂ SO4. andby stirring overnight at 4° C. The precipitate was centrifuged at 14,000g for 10 min at 4° C. and the pellet was washed in an equal volume ofcold 50% (NH4)₂ SO4. The precipitate was centrifuged again at 14,000 gfor 10 min at 4° C., dissolved in PBS without calcium or magnesium, pH7.5, and dialyzed extensively in PBS to remove all (NH4)₂ SO4. Purity ofthe IgY preparation was confirmed by SDS-PAGE (approx. 90-95%), andquantitation of IgY was estimated by measuring the absorbance at 280 nmand using an extinction coefficient of 1.3.

Affinity purification of IgY from chickens injected with the primarypeptide of SEQ ID NO:8 was accomplished by first covalently linking thepeptide to SULFOLINK coupling gel (Pierce Chemical Company), whichallows binding specifically to sulfhydryl groups such as that on theC-terminal cysteine of the peptide. A 3 ml column was made with 3 mg ofpeptide according to the product instructions. Briefly, a 3 ml columnwas equilibrated with 6 column volumes of 50 mM Tris, 5 mM EDTA, pH 8.5,and then 3 mg of the primary peptide SEQ ID NO:8 in 3 ml 50 mM Tris, 5mM EDTA pH 8.5 were added to the column for mixing at room temperaturefor 15 min. The column gel and peptide were incubated for another 30 minwithout mixing. The peptide buffer was drained off the gel and saved forlater testing to confirm coupling efficiency using Ellman's reagent(DTNB (5,5'-dithiobis(2-nitrobenzoic acid), Pierce Chemical Company)which detects sulfhydryl groups. The primary peptide SEQ ID NO:8 doesnot contain any aromatic amino acid groups and could not be detectedspectrophotometrically or by standard protein assay techniques, such asby Bradford analysis. Using Ellman's reagent according to the productinstructions for comparison of an aliquot of peptide solution before andafter binding to the gel, confirmed 100% binding efficiency. The gelcolumn was washed with 3 column volumes of 50 mM Tris, 5 mM EDTA pH 8.5before blocking nonspecific binding sites with 3 ml of cysteine solutionin 50 mM Tris, 5 mM EDTA pH 8.5 for 15 min mixing at room temperaturefollowed by 30 minutes without mixing. The column was drained, andwashed with 16 column volumes of 1M NaCl and then with 16 column volumesof degassed 0.05% sodium azide.

IgY was affinity purified on this peptide-linked SULFOLINK gel accordingto a modified version of Rosol et al. Veterinary Immunology andImmunopathology, 35:321-337, 1993. Once at room temperature, the columnwas washed with 10 column volumes of PBS. IgY was recycled on the columnfor 2 hours. The column was then washed with 10 column volumes of PBSfollowed by 10 column volumes of phosphate buffered saline (PBS) with0.5M NaCl. Peptide-specific IgY was eluted with 500 mM glycine pH 2.5and neutralized with 1M Tris pH 9.5. A UV spectrophotometer and graphingapparatus were used to follow the washing and elution of protein off thecolumn. Samples with a signal at OD280 nm were concentrated in acentriprep 30 (Amicon) to a volume of 500-600 μl.

Fab fragments ("Yab fragments") were made from affinity purified IgYincubated with immobilized pepsin (Pierce Chemical Company) according toproduct instructions and modified from the method of Akita and Nakai.Journal of Immunological Methods. 162:155-164, 1993. Pepsin slurry waswashed twice with 16 times the volume of 50 mM sodium acetate buffer pH4.2, and resuspended in twice the volume of sodium acetate buffer.Affinity purified IgY was incubated with the immobilized pepsin at 37°C. and mixed for 5 hour. One molar Tris-HCI pH 8.0 was added to give afinal pH of 7.5. The pepsin mixture was centrifuged at 1000 g for 5 minand the supernatant containing the fragments was added to a CENTRICON 10filter (Amicon) to remove small Fc fragments. Complete cleavage wasconfirmed by SDS-PAGE.

Chicken serum from successive test bleeds and IgY extracted from batchesof pooled egg yolks were tested by ELISA to confirm recognition of thepeptide. Affinity purified IgY and Fab' fragments ("Yab") were testedfor their ability to recognize intact pIgR by western blot. Cell lysateswere made from Madin-Darby canine kidney (MDCK) cells and MDCK cellstransfected with rabbit pIgR ("pWe"), according to the method ofBreitfeld et al. (Methods in Cell Biology 32:329-337 (1989)) using 10%NP40 lysis buffer containing 1 μg/ml of protease inhibitors andphenylmethylsulfonyl fluoride (PMSF). Cell lysates were run on a 10% gelunder reducing conditions and transferred onto a PVDF(polyvinyldifluoride) membrane (Millipore, Bedford, Mass.). A mousemonoclonal antibody to the cytoplasmic portion of pIgR, SC 166 (Solariet al., Cell, 36:61-71 (1984)), was used as a positive control antibody,and IgY isolated from pre-immune yolks was used as a negative control.HRP-conjugated rabbit anti-chicken IgY (Jackson Immunochemicals) andHRP-conjugated rabbit anti-mouse (Biorad) were used as secondaryantibodies. IgY from a chicken injected with the primary peptide and IgYfrom a chicken injected with the subsequent peptide recognized intactpIgR, but IgY from one of the chickens injected with the subsequentantibody did not. Immunofluorescence studies of IgY and Yab fragments(from chickens injected with the primary peptide) with MDCK and pWecells grown on coverslips, fixed with 4% paraformaldehyde andpermeabilized with saponin showed more specific staining of thepIgR-transfected cells (FITC-conjugated rabbit anti-chicken andanti-mouse antibodies obtained from Jackson Immunochemicals). A cellELISA (modified from M Hahne et al., Journal of Cell Biology.121:655-64, 1993) on fixed and permeabilized cells showed Yab fragmentstaining 5-fold greater with pWe cells than MDCK cells. These datademonstrate that we have successfully raised polyclonal antibodiesagainst the rabbit pIgR stalk peptide and that they recognize intactpIgR.

EXAMPLE 2

Example 2 describes selection of human recombinant single chain variableregion fragment (scFv) antibodies by phage display.

Selection of scFv by phage display requires a soluble biotinylatedantigen or antigen immobilized on a solid support. Because scFv selectedby phage display tend to be low affinity binders and because the solubleantigen may allow selection of higher affinity scFv (R Schier et al., J.Mol. Biol. 255:28-43, 1996), the selection approach with soluble antigenwas chosen. The pIgR stalk primary peptide, described in Example 1,corresponding to the 23 amino acids of the membrane-proximalextracellular part of the rabbit pIgR was conjugated to biotin via thesulfhydryl group of the cysteine residue using biotin-BMCC((1-Biotinamido-4-(4'[maleimidomethyl]cyclohexane-carboxamido) butane)(Pierce Chemical Company, Rockford, Ill.) based on the method describedin the product instructions. To ensure that the peptide had notdimerized via the sulfhydryl groups, the peptide was first reduced with1% sodium borohydride in 0.1M Tris, 5 mM EDTA pH 8.0. The pH of thesolution was lowered to pH 5 by adding 1N HCl. Once the solution hadfinished fizzing, 1M Tris was added back to reach pH 7.0. A 8.5 mMbiotin-BMCC solution was prepared by dissolving the biotinylationreagent in DMSO. A 5-fold molar excess of biotin-BMCC was added to thereduced peptide and incubated overnight at 4° C. The biotinylatedpeptide was separated from free biotin by HPLC with a C18 column with agradient ranging from 10 to 50% CH₃ CN over 30 min, with UV detection at215 nm. Mass spectrometry by electrospray and LSIMS (liquid secondaryion mass spectrometry) identified the correct peak corresponding to thebiotinylated peptide.

The biotinylated primary peptide was incubated with a phage libraryencoding a large number of different human scFv (approx. 10¹⁰). Thisphage library was prepared as previously described (J D Marks et al., J.Mol. Biol. 222:581-97, 1991; J D Marks et al., Bio/Technology10;779-783, 1992; J D Marks et al., Bio/Technology 11:1145-1149, 1993; AD Griffiths et al., EMBO J 12:725-734, 1993). A total of four rounds ofselection, phagemid rescue and expansion in Escherichia coli suppressorstrain TG-1 were performed as described in Marks et al. (J. Mol. Biol.222:581-97, 1991) with the following modifications. The phage libraryused is known to contain several streptavidin binders, so the firstthree rounds of selection included a preclearing step with two 30 minincubations of the phage with streptavidin agarose (Sigma). The phagewere then incubated with 5 μg of biotinylated primary peptide for 1hour. To bind the biotinylated peptide with the attached phage, thepeptide-phage solution was incubated with avidin magnetic beads on thefirst and third rounds for 15 and 5 minutes, respectively, and withstreptavidin magnetic beads on the second and fourth rounds for 10 and 5minutes, respectively. Rescued phage from the fourth round of selectionwere infected into Escherichia coli non-suppressor strain HB2151, andindividual phagemid clones were induced to produce soluble scFvfragments with IPTG as described in Marks et al. (J. Mol. Biol.222:581-97, ((1991)).

Bacterial supernatants from the individual clones were analysed forexpression of soluble scFv fragments in a dot blot assay and for bindingto biotinylated primary peptide in an ELISA assay (Finnern et al., Clin.Exp. Immunol. 102:566-574, 1995). The ELISA assay, however, was modifiedin the following manner: 96-well microwell plates (Immulon-4) werecoated with avidin (10 μg/ml in phosphate buffered saline (PBS))overnight at 4° C., washed 3 times with PBS, blocked with 2% milk in PBSand bound with biotinylated primary peptide (5 μg/ml in PBS). TMB(3,3',5,5' tetramethylbenzidine) solution (Kirkegaard and Perry) wasused as substrate (100 μl/well), and the reaction was stopped with 0.18MH₂ SO4 before reading the color reaction in an ELISA reader at awavelength of 450 nm. Dot blot analysis showed that 66% of the 96selected colonies of HB2151 infected with phage rescued from the fourthround of selection produced scFv. ELISA assay showed that 43 of the 96colonies produces scFv that bound to the peptide.

The diversity of all positive clones was determined by PCR screening.The scFv insert of the heavy and light chain was first amplified withthe primers LMB3 and fd-Seq1 (Marks et al., J. Mol. Biol. 222:581-97,1991), and then digested with the restriction enzyme BstN1. Clones withdifferent DNA fingerprint patterns were sequenced using a SequiThermLong-Read cycle sequencing kit (Epicentre Technologies) and a Licormachine. Five unique sequences were identified.

To obtain large amounts of purified scFv for further characterizationand use, the five unique scFv were subcloned into the expression vectorpUC119 Sfi-NotmycHis, which adds a hexa-histidine tag at the C-terminalend of the scFv (Schier et al., J. Mol. Biol., 255:28-43, 1996).

EXAMPLE 3

Example 3 describes targeting of the wildtype cystic fibrosistransconductance regulator (CFTR) gene into mammalian cells expressingpIgR using a variation of the methods disclosed in Ferkol et al., J.Clin. Invest., 92:2394-2400 (1993); and Ferkol et al., J. Clin. Invest.,95:493-502 (1995), each of which is incorporated herein by reference.

An Fab fragment reactive to the stalk region of pIgR is made andpurified by techniques such as that disclosed in Example 1. The Fab islinked to poly (L-lysine) (MW 20,000 Daltons) using theheterobifunctional crosslinking reagent N-succinimidyl3-(2-pyridyidithio) propionate (SPDP) according to the method of Ferkolet al. (1993).

A plasmid comprising the CFTR gene is ligated to a cytomegalovirus earlypromoter and inserted into the vector pCB6. Thomas et al., J. Biol.Chem., 268:3313-3320 (1993). Complexes of Fab-polylysine-DNA are made bycombining plasmid DNA with the Fab-polylysine in 3M NaCl.

The complex is introduced by dissolving it in 0.1 ml of phosphatebuffered saline, and placing it into the nares of pathogen-freeSprague-Dawley rats (250-300 grams) lightly anesthetized with Metofaneinhalant anesthesia. A micropipet will be used to apply 100 μL of theplasmid in PBS directly into the nares of rats that are manuallyrestrained in the supine position. Rats will be held in this positionuntil the solution has been inhaled. This technique has been shown toresult in effective application of the sample onto the nasal mucosa.Shahin et al., Infection and Immunity 60:1482-1488 (1992); Gizurarson etal., Vaccine 10:101-106 (1992). Transcription of the transfected gene isassayed by immunofluorescence assay of production of the CFTR protein.

EXAMPLE 4

Example 4 describes a means of in vivo targeting of exogenous proteinsinto cells expressing pIgR.

An efficient method to allow egress of proteins from endosomes willemploy the protein-Fab complex coupled to adenovirus. This method hasbeen used with a number of receptor systems resulting in as much as a1000-fold increase in expression. Curiel et al., J. Respir. Cell Mol.Biol. 6:247-252 (1992); Curiel et al., Proc. Natl. Acad. Sci. USA88:8850-8854 (1991), Gao et al., Hum. Gene Ther. 4:17-24 (1993), each ofwhich is incorporated herein by reference. Coupling is accomplished bybiotinylation of the adenovirus and the Fab/poly-lysine followed bycross-linking with avidin. The resultant complex is administered as inExample 3.

EXAMPLE 5

Example 5 describes transcytosis of antibodies, which specifically bindto the stalk, from the apical to basolateral membrane of a MDCK (MadinDarby canine kidney) cell comprising pIgR.

Yab fragments reactive to pIgR were made as described in Example 1.Anti-pIgR stalk Yab fragments were radio-iodinated by the iodinemonochloride method of Goldsein et al. (Meth. Enzymol. 96:241-249,1983). Radio-iodinated IgA was used as a control ligand. Radio-iodinatedYab fragments or IgA (10⁷ cpm in 100 μl /well) were added to the apicalsurface of MDCK and pWe cells grown in a polarized manner for 4 days on12 mm diameter, 0.4 μm pore size cell culture inserts (Transwells,Costar). Breitfeld et al., Methods in Cell Biology 32:329-337 (1989).Radio-labelled Yab fragments were added to cells with and without a 2 hpreincubation with the protease inhibitor, leupeptin, 50 μl/ml. After 20min of apical uptake at 37° C., unbound radio-labelled ligand was washedwith MEM (minimal essential medium)/BSA (bovine serum albumen) threetimes quickly, one 5 min wash and two more quick washes. The apical andbasolateral media were collected and changed at 7, 15, 30, 60 and 120min time points for quantitation in a gamma counter (BeckmanInstruments, Palo Alto, Calif.). Cell culture inserts were cut out at120 min for quantitation in a gamma counter and to calculate the totalinitial uptake of radioactivity. The background uptake by MDCK cells wassubtracted from that by pWe cells to calculate specific recycling andtranscytosis of the radiolabeled ligands. The results showed that 13-18%of the Yab fragments were transcytosed from the apical to thebasolateral surface of cells pretreated with leupeptin, in contrast to5-6% of the IgA.

All publications and patents mentioned in this specification are hereinincorporated by reference into the specification to the same extent asif each individual publication or patent was specifically andindividually indicated to be incorporated herein by reference.

    __________________________________________________________________________    #             SEQUENCE LISTING                                                - (1) GENERAL INFORMATION:                                                    -    (iii) NUMBER OF SEQUENCES: 11                                            - (2) INFORMATION FOR SEQ ID NO:1:                                            -      (i) SEQUENCE CHARACTERISTICS:                                          #acids    (A) LENGTH: 4 amino                                                           (B) TYPE: amino acid                                                          (C) STRANDEDNESS:                                                             (D) TOPOLOGY: linear                                                -     (ii) MOLECULE TYPE: peptide                                             -     (ix) FEATURE:                                                                     (A) NAME/KEY: Modified-sit - #e                                               (B) LOCATION: 3                                                     #/product= "OTHER"R INFORMATION:                                              #"Xaa = polar or charged amino                                                               acid"                                                          -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:1:                                 - Phe Ala Xaa Glu                                                             - (2) INFORMATION FOR SEQ ID NO:2:                                            -      (i) SEQUENCE CHARACTERISTICS:                                          #acids    (A) LENGTH: 33 amino                                                          (B) TYPE: amino acid                                                          (C) STRANDEDNESS:                                                             (D) TOPOLOGY: linear                                                -     (ii) MOLECULE TYPE: peptide                                             -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:2:                                 - Glu Arg Glu Ile Gln Asn Val Arg Asp Gln Al - #a Gln Glu Asn Arg Ala         #                15                                                           - Ser Gly Asp Ala Gly Ser Ala Asp Gly Gln Se - #r Arg Ser Ser Ser Ser         #            30                                                               - Lys                                                                         - (2) INFORMATION FOR SEQ ID NO:3:                                            -      (i) SEQUENCE CHARACTERISTICS:                                          #acids    (A) LENGTH: 31 amino                                                          (B) TYPE: amino acid                                                          (C) STRANDEDNESS:                                                             (D) TOPOLOGY: linear                                                -     (ii) MOLECULE TYPE: peptide                                             -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:3:                                 - Glu Arg Glu Ile Gln Asn Ala Gly Asp Gln Al - #a Gln Glu Asn Arg Ala         #                15                                                           - Ser Gly Asn Ala Gly Ser Ala Gly Gly Gln Se - #r Gly Ser Ser Lys             #            30                                                               - (2) INFORMATION FOR SEQ ID NO:4:                                            -      (i) SEQUENCE CHARACTERISTICS:                                          #acids    (A) LENGTH: 31 amino                                                          (B) TYPE: amino acid                                                          (C) STRANDEDNESS:                                                             (D) TOPOLOGY: linear                                                -     (ii) MOLECULE TYPE: peptide                                             -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:4:                                 - Glu Lys Ala Val Ala Asp Thr Arg Asp Gln Al - #a Asp Gly Ser Arg Ala         #                15                                                           - Ser Val Asp Ser Gly Ser Ser Glu Glu Gln Gl - #y Gly Ser Ser Arg             #            30                                                               - (2) INFORMATION FOR SEQ ID NO:5:                                            -      (i) SEQUENCE CHARACTERISTICS:                                          #acids    (A) LENGTH: 27 amino                                                          (B) TYPE: amino acid                                                          (C) STRANDEDNESS:                                                             (D) TOPOLOGY: linear                                                -     (ii) MOLECULE TYPE: peptide                                             -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:5:                                 - Glu Ser Val Lys Asp Ala Ala Gly Gly Pro Gl - #y Ala Pro Ala Asp Pro         #                15                                                           - Gly Arg Pro Thr Gly Tyr Ser Gly Ser Ser Ly - #s                             #            25                                                               - (2) INFORMATION FOR SEQ ID NO:6:                                            -      (i) SEQUENCE CHARACTERISTICS:                                          #acids    (A) LENGTH: 40 amino                                                          (B) TYPE: amino acid                                                          (C) STRANDEDNESS:                                                             (D) TOPOLOGY: linear                                                -     (ii) MOLECULE TYPE: peptide                                             -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:6:                                 - Leu Ala Glu Val Ala Val Gln Ser Ala Glu As - #p Pro Ala Ser Gly Asp         #                15                                                           - Pro Ala Ser Gly Ser Arg Ala Ser Val Asp Se - #r Gly Ser Ser Glu Glu         #            30                                                               - Gln Gly Gly Ser Ser Arg Ser Lys                                             #        40                                                                   - (2) INFORMATION FOR SEQ ID NO:7:                                            -      (i) SEQUENCE CHARACTERISTICS:                                          #acids    (A) LENGTH: 23 amino                                                          (B) TYPE: amino acid                                                          (C) STRANDEDNESS:                                                             (D) TOPOLOGY: linear                                                -     (ii) MOLECULE TYPE: peptide                                             -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:7:                                 - Asp Pro Ala Ser Gly Ser Arg Ala Ser Val As - #p Ala Ser Ser Ala Ser         #                15                                                           - Gly Gln Ser Gly Ser Ala Lys                                                             20                                                                - (2) INFORMATION FOR SEQ ID NO:8:                                            -      (i) SEQUENCE CHARACTERISTICS:                                          #acids    (A) LENGTH: 24 amino                                                          (B) TYPE: amino acid                                                          (C) STRANDEDNESS:                                                             (D) TOPOLOGY: linear                                                -     (ii) MOLECULE TYPE: peptide                                             -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:8:                                 - Asp Pro Ala Ser Gly Ser Arg Ala Ser Val As - #p Ala Ser Ser Ala Ser         #                15                                                           - Gly Gln Ser Gly Ser Ala Lys Cys                                                         20                                                                - (2) INFORMATION FOR SEQ ID NO:9:                                            -      (i) SEQUENCE CHARACTERISTICS:                                          #acids    (A) LENGTH: 17 amino                                                          (B) TYPE: amino acid                                                          (C) STRANDEDNESS:                                                             (D) TOPOLOGY: linear                                                -     (ii) MOLECULE TYPE: peptide                                             -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:9:                                 - Ala Ser Val Asp Ala Ser Ser Ala Ser Gly Gl - #n Ser Gly Ser Ala Lys         #                15                                                           - Cys                                                                         - (2) INFORMATION FOR SEQ ID NO:10:                                           -      (i) SEQUENCE CHARACTERISTICS:                                          #acids    (A) LENGTH: 61 amino                                                          (B) TYPE: amino acid                                                          (C) STRANDEDNESS:                                                             (D) TOPOLOGY: linear                                                -     (ii) MOLECULE TYPE: peptide                                             -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:10:                                - Ala Asp Ala Ala Pro Asp Glu Lys Val Leu As - #p Ser Gly Phe Arg Glu         #                15                                                           - Ile Glu Asn Lys Ala Ile Gln Asp Pro Arg Le - #u Phe Ala Glu Glu Lys         #            30                                                               - Ala Val Ala Asp Thr Arg Asp Gln Ala Asp Gl - #y Ser Arg Ala Ser Val         #        45                                                                   - Asp Ser Gly Ser Ser Glu Glu Gln Gly Gly Se - #r Ser Arg                     #    60                                                                       - (2) INFORMATION FOR SEQ ID NO:11:                                           -      (i) SEQUENCE CHARACTERISTICS:                                          #acids    (A) LENGTH: 61 amino                                                          (B) TYPE: amino acid                                                          (C) STRANDEDNESS:                                                             (D) TOPOLOGY: linear                                                -     (ii) MOLECULE TYPE: peptide                                             -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:11:                                - Ala Asp Ala Ala Pro Asp Glu Lys Val Leu As - #p Ser Gly Phe Arg Glu         #                15                                                           - Ile Glu Asn Lys Ala Ile Gln Asp Pro Arg Le - #u Phe Ala Glu Glu Lys         #            30                                                               - Ala Val Ala Asp Thr Arg Asp Gln Ala Asp Gl - #y Ser Arg Ala Ser Val         #        45                                                                   - Asp Ser Gly Ser Ser Glu Glu Gln Gly Gly Se - #r Ser Arg                     #    60                                                                       __________________________________________________________________________

What is claimed is:
 1. A method of introducing a ligand into a cellexpressing a polymeric immunoglobulin receptor by binding the ligand tothe stalk of the polymeric immunoglobulin receptor (pIgR) of the cell atthe cell's apical surface with the proviso that the ligand does notsubstantially bind to the secretory component of pIgR underphysiological conditions.
 2. A method of claim 1 wherein the ligand isan antibody.
 3. A method of claim 1 wherein the ligand is a humanizedantibody.
 4. A method of claim 2 wherein the ligand is a recombinantsingle chain variable region fragment of an antibody.
 5. A method ofclaim 1 wherein the ligand binds to an extracellular epitope within thefirst 33 amino acids that are cell membrane proximal to a cleavage siteof the receptor.
 6. A method of claim 1 wherein the ligand binds to apeptide selected from the group of:Mouse:Glu-Arg-Glu-Ile-Gln-Asn-Val-Arg-Asp-Gln-Ala-Gln-Glu-Asn- (SEQ IDNO:2)Arg-Ala-Ser-Gly-Asp-Ala-Gly-Ser-Ala-Asp-Gly-Gln-Ser-Arg-Ser-Ser-Ser-Ser-LyRat:Glu-Arg-Glu-Ile-Gln-Asn-Ala-Gly-Asp-Gln-Ala-Gln-Glu-Asn-Arg- (SEQ IDNO:3)Ala-Ser-Gly-Asn-Ala-Gly-Ser-Ala-Gly-Gly-Gln-Ser-Gly-Ser-Ser-LysHuman:Glu-Lys-Ala-Val-Ala-Asp-Thr-Arg-Asp-Gln-Ala-Asp-Gly-Ser- (SEQ IDNO:4)Arg-Ala-Ser-Val-Asp-Ser-Gly-Ser-Ser-Glu-Glu-Gln-Gly-Gly-Ser-Ser-ArgBovine:Glu-Ser-Val-Lys-Asp-Ala-Ala-Gly-Gly-Pro-Gly-Ala-Pro-Ala- (SEQ IDNO:5)Asp-Pro-Gly-Arg-Pro-Thr-Gly-Tyr-Ser-Gly-Ser-Ser-LysRabbit:Leu-Ala-Glu-Val-Ala-Val-Gln-Ser-Ala-Glu-Asp-Pro-Ala-Ser- (SEQ IDNO:6)Gly-Asp-Pro-Ala-Ser-Gly-Ser-Arg-Ala-Ser-Val-Asp-Ser-Gly-Ser-Ser-Glu-Glu-Gln-Gly-Gly-Ser-Ser-Arg-Ser-Lys.7. A method of claim 1 wherein the ligand is further defined as having abinding component for selectively binding to the receptor stalk and abiologically active component wherein the biologically active componentis selected from the group consisting of a nucleic acid, protein,radioisotope, lipid, and carbohydrate.
 8. A method of claim 1 whereinthe cell is a mammalian cell.
 9. A method of claim 8 wherein the cell isan epithelial cell.
 10. The method of claim 1 wherein the ligand istranscytosed to the basolateral side of the cell.
 11. The method ofclaim 10, wherein the ligand is released from the stalk at thebasolateral surface of the cell.
 12. The method of claim 1 wherein theligand specifically binds only to the stalk.
 13. A method of binding aligand to a cell having an apical surface and expressing a polymericimmunoglobulin receptor (pIgR) comprising the step of binding the ligandto a stalk of the receptor at the apical surface with the proviso thatthe ligand does not substantially bind to secretory component of pIgRunder physiological conditions.
 14. The method of claim 13 wherein theligand is endocytosed into the cell after binding.